The central nervous system regulates the reproductive axis through the activity of the "GnRH pulse generator", a set of neurons whose activity governs pulsatile release of gonadotropin-releasing hormone (GnRH). The amplitude and frequency of GnRH pulses are, in turn, regulated by important physiological signals, such as gonadal hormones. Progesterone (P) is one important regulator that suppresses pulsatile GnRH release, however the cellular bases of these negative feedback actions remain largely unknown. These studies are designed to determine how P exerts negative feedback actions on the GnRH pulse generator, and how sensitivity of the GnRH pulse generator to P's suppressive effects may be regulated. In Aim 1 we will test the hypothesis that the intracellular progesterone receptor (PR) isoforms, PRA and PRB, mediate P negative feedback actions. A microdialysis approach will be used to determine if specific PR receptor antagonism can block P-mediated suppression of GnRH pulsatility in vivo. In Aim 2, we will attempt to identify the neural locus of P's inhibitory actions. PR antisense oligonucleotides will be used to "knockdown" PRA and PRB expression in different neural regions and any reductions in sensitivity to P will be assessed. Experiments in Aims 3 & 4 will test the hypothesis that at least some PRs mediating P inhibitory actions are expressed in GnRH neurons themselves. A single-cell PCR method will be used in tissue slices from GnRH-GFP-tagged transgenic mice (Aim 3) to determine whether PR mRNA is expressed in identified GnRH neurons in situ. Cell-specific gene targeting will then be used (Aim 4) to determine if PR expression in GnRH neurons mediates P negative feedback; mice bearing a GnRH neuron-specific deletion of the PR gene will be generated and then tested for resistance to P's negative feedback effects. Experiments in Aim 5 focus on mechanisms by which responsiveness to P negative feedback may be altered under pathophysiological conditions. We will specifically test the hypothesis that P's actions on the GnRH pulse generator are downregulated by androgens, through a mechanism involving androgen suppression of neuronal PR expression. These studies will provide new and important information on cellular mechanisms mediating homeostatic regulation of the female reproductive axis. They may also permit a new understanding of the neuroendocrine basis of GnRH hyperpulsatility that is characteristic of conditions such as polycystic ovarian syndrome.